HKT1 encodes a high affinity Na+ coupled K+ transporter expressed in the cortical cells of Triticum aestivum roots. To identify regions of the protein involved in the binding and transport of Na+ and K+, mutations were introduced into a domain of HKT1 containing 16 amino acids that are highly conserved across a range of putative K+ transport proteins from different phyla. Two mutations had a significant effect on the functional characteristics of the transporter. A yeast growth assay showed that concentrations of NaCl between 2.5 to 50 mM stimulated the growth of yeast expressing HKT1 containing the E464Q substitution, but not the growth of yeast expressing HKT1. Kinetic analysis confirmed that the E464Q mutation lowered the affinity of HKT1 for Na+ but did not affect its affinity for K+. A second mutation in the same region F463L was created that also lowered the affinity of the transporter for Na+. The importance of these highly conserved amino acid residues is highlighted by the fact that they have remained conserved through evolution. The results of this mutational analysis suggest that this domain in HKT1 plays a role in the binding and transport of Na+.